Leptin favors imbalance of antigen-specific CD4+ T-cells associated with severity of cat allergy

Introduction Obesity can complicate IgE-mediated allergic diseases. In the present study, we aimed to investigate the ability of obesity-related concentrations of leptin to modulate the in vitro effector and regulatory Fel d1-specific CD4+ T-cell subsets in patients allergic to cat, considered the third most common cause of respiratory allergy in humans. Methods For this study, plasma and peripheral blood mononuclear cells (PBMC) from 30 cat-allergic patients with mild, moderate and severe respiratory symptoms were obtained. The PBMC cultures were stimulated with Fel d1 antigen (10 µg/mL) in the presence or absence of obesity-related leptin dose (50 ηg/mL). After 6 days, the levels of cytokines and IgE in the supernatants were evaluated by multiplex and ELISA, respectively. The frequency of different non-follicular (CXCR5-) and follicular (CXCR5+) Fel d1-specific CD4+ T cell subsets was determined by flow cytometry. The plasma levels of leptin and IgE anti-cat titers were evaluated by ELISA and ImmunoCAP, respectively. Results and conclusions Fel d1 induced both IgE production and release of cytokines related to Th2, Th9 and Th17 cell phenotypes. Feld1 was more efficient in increasing the frequency of TFHIL-21- cells positive for IL-4, IL-5 and IL-13 than TFHIL-21+ cell subsets. Leptin favored the expansion Th2-like and Th9-like cells and TFHIL-21- cells positive for IL-4, IL-5 and IL-13, but reduced the proportion of conventional (Treg/Tr-1) and follicular (TFR) regulatory CD4+ T-cell subsets expressing or not CD39 marker. Finally, many of the imbalances between Fel d1-specific CD4+ T-cells were also correlated with plasma leptin and anti-Fel d1 IgE titers. In summary, hyperleptinemia should negatively impact on the severity of cat allergies by favoring the expansion of pathogenic Fel d1-specific CD4+ T-cell phenotypes and damaging the functional status of regulatory CD4+ T-cell subsets.


Introduction
Cat allergies are the most common mammalian-origin allergy in humans, affecting approximately 1 in 5 adults worldwide (1,2).The most common clinical presentations in these patients are rhinitis, asthma, and/or conjunctivitis.When persistent, the clinical symptoms may impair quality of life (3,4).Furthermore, severely allergic patients may present an anaphylactic reaction, requiring emergency medical care.Although eight allergens derived from cats have been described, designated Fel d1 to d8, only Fel d1 has clinical significance, accounting for up to 96% of allergic sensitization to cats in humans (5,6).Primarily produced by salivary and sebaceous glands (5).Fel d1 can easily become and remain airborne in dander and dust particles for extended periods (5,6).
The hallmark of cat sensitization and symptom severity is the production of high-affinity Fel d1-specific IgE (6,7).Although Th2 cytokines, IL-4 and IL-13, can increase IgE production, the synthesis of this antibody is critically dependent on B cell collaboration with follicular helper T (T FH ) cells (8).T FH cells are specialized CD4 + T cells that provide help to B cells activation into germinal center (GC) of lymphoid follicles.In the GC, T FH cells are characterized by high expression of CXCR5, programmed cell death protein (PD-1), B cell lymphoma 6 (Bcl-6), and IL-21 production (9).The main function of CXCR5 is to guide T FH cells migration towards lymphoid follicles in response to its ligand, the CXCL13, abundantly produced by GC-derived B cells (9).On the other hand, IL-21 from T FH cells not only mediate the selection of high-affinity and isotype switched B cells, but also promote differentiation of these lymphocytes into plasma cells and memory B cells (9).Although T FH cells in peripheral blood are Bcl-6 negative and express low PD-1 levels, they are able to induce antibody production from peripheral B cells (9).Based on cytokines, human circulating T FH cells have been classified as T FH 1 (IL-21 + IFN-g + ), T FH 2 (IL-21 + IL-4 + ), T FH 17 (IL-21 + IL-17 + ) and, more recently, T FH 13 (IL-21 low L-4 hi IL-5 hi IL-13 hi ) (8).
Many studies have demonstrated the involvement of T FH 2 cells in the pathogenesis of allergic IgE-mediated airway diseases (10)(11)(12)(13)(14)(15)(16).In patients suffering from allergic rhinitis and asthma, elevated frequency of circulating T FH 2 has been associated with plasma IgE titers and clinical exacerbation (10-14).Moreover, the expansion of T FH 2 cells inside the airways of allergic patients appear to promote IgE production by local activated B cells, which may play an important role in mast cells and eosinophil activation (15,16).
Interestingly, more recent studies have demonstrated that, while T FH 2 cells induce low-affinity IgE production, the synthesis of highaffinity IgE to allergens critically depends on T FH 13 cells (8,17).The binding of high-affinity IgE/allergen to FceRI on mast cells and basophils immediately triggers histamine release, quickly causing a cluster of typical cat allergic symptoms (18).Further, in addition to mast cells, eosinophils activated by the same Fel d1/IgE complexes contribute to allergy pathogenesis by producing lager amounts of leukotrienes (LTC4, LTD4, and LTE4) and platelet-activating factor (PAF), pro-inflammatory lipids that induce local vasodilatation, edema, neurogenic stimulation, smooth muscle contraction and hypersecretion of mucus (19).Moreover, IL-9-secreting CD4 + T (Th9) cells have also been implicated in atopic allergy (20).IL-9 prolongs the survival of mast cells, potentiates IgE production and amplifies the ability of IL-5 and IL-13 to increase eosinophil survival and mucus production (20).
Obesity has been related to severity of allergy symptoms and to higher levels of total and allergen-specific IgE in atopic individuals (23,24).In cat allergic patients, obesity was associated with total and allergen-specific IgE levels (25).This adverse relationship must be, at least partially, associated with high leptin production, an adipokine known to modulate the functional status of T cells (26).
Leptin is a 16 kDa peptide encoded by the OB gene.At physiological concentrations, leptin plays an adjuvant role in the immune response against different pathogens (27).However, hyperleptinemia, as observed in obese individuals, has been correlated with the severity of allergic reactions (28).Ciprandi et al. (29) demonstrated a direct relationship between IgE titers and eosinophil counts with leptin levels in patients with allergic rhinitis.With regard to CD4 + T cell phenotypes, studies published by our group performed in patients with allergic asthma (AA) have found a positive correlation between plasma leptin levels and circulating Th2and Th17-like cells able to produce high levels of IL-5, IL-6 and IL-17 in response to mitogen (30,31).In addition, the frequency of these pro-inflammatory CD4 + T cell subsets were directly associated with lung function impairment (31).Still according to our previous study, in CD4 + T cell cultures from lean AA patients, obesity-related leptin concentration enhanced Th2-and Th17-related cytokine production and impaired Treg function in response to polyclonal activators (31).However, studies regarding the effects of leptin on the composition of different allergen-specific CD4 + T-cells have not been conducted to date.Therefore, the main objective of the present study was to investigate the ability of obesity-related leptin doses to modulate the in vitro different effector and regulatory Fel d1-specific CD4 + T cells from patients with persistent cat allergies.
According to daily frequency, severity of clinical exacerbation, lung function damage and need to hospital admission, AA is classified as mild, moderate or severe (21).Also, interference in daily activities is also taken into account (21).With regard to AR, symptom severity was determined by using the total nasal symptom score (TNSS), which is calculated as the sum of scores for each of nasal congestion, sneezing, nasal itching, and rhinorrhea at each time point, using a four point scale (0-3), where 0 indicates no symptoms, 1 for mild symptoms, 2 for awareness of symptoms (but tolerable), and score 3 for severe symptoms that are hard to tolerate and interfere with daily activity (32) (Table 1).We excluded patients taking oral or intravenous steroids, theophylline, long-acting b2-agonists, leukotriene antagonists or antihistamines 1 month prior to the study.As control group, twenty healthy subjects (HS), matched for age and sex and with no history of allergic diseases, were also recruited into the study.
According to the body mass index (BMI), subjects were stratified as lean (BMI from 18.5 to 24.9), overweight (BMI from 25 to 29.9) and class I obesity (BMI from 30 to 35

Cell cultures
Peripheral blood was collected in heparin-containing tubes (BD Vacutainer, Franklin Lakes, NY) and peripheral blood mononuclear cells (PBMC) were obtained by centrifugation on the Ficoll-Hypaque density gradient.Fresh viable PBMCs (1 × 10 6 /mL) were cultured in 24-well flat-bottomed microplates with 2 mL of RPMI medium (ThermoFisher Scientific Inc.) supplemented with 2 mM of L-glutamine (GIBCO, Carlsbad, CA, USA), 10% fetal calf serum, 20 U/mL of penicillin, 20 mg/mL of streptomycin and 20 mM of HEPES buffer.As positive control, PBMC cultures were stimulated with phytohaemaglutinine (PHA, 1 µg/mL) (Sigma-Aldrich Co) for 3 days in a humidified 5% CO 2 incubator.In order to evaluate the antigen-specific response, the cells were stimulated with Fel d1 (10 µg/mL) (MyBioSource, San Diego, CA, USA) for 6 days.This concentration of Fel d1 was chosen from a previous study that evaluated T cell response to this antigen (6).In these cultures, the role of leptin (Sigma-Aldrich Co) was determined after the addition of 50 ng/mL of this adipokine.This leptin concentration was determined after a dose-response curve (10, 50 and 100 ng/mL) of cytokine-secreting CD4 + T cells from healthy subjects (HS) and cat-allergic patients (CAP) in PBMC cultures activated with PHA (1 µg/mL) (Sigma-Aldrich Co) (Figure S1).Notably, this leptin concentration is related to the levels of this adipokine in obese subjects (33).After 6 days of culturing, the supernatants were collected, frozen at -20°C for further analysis of cytokine production (Luminex) and IgE levels (ELISA).The PBMC Healthy subjects. 2Cat allergic patients suffering from rhinitis, asthma alone or rhinitis and asthma to cat dander. 3The severity of rhinitis and asthma symptom was determined by TNSS (total nasal symptom score) and GINA (Global Initiative for Asthma) criteria, respectively. 4Body mass index: a value derived from the mass (weight in Kg) and height (in meters) of an individual (lean: 18.5-24.9,overweight: 25-29.9 and obese class I: [30][31][32][33][34][35].ND, no determined.
was also used to identify different CD4 + T cell phenotypes using flow cytometry.

Luminex, ImmunoCAP and ELISA assays
The titers of plasma IgE anti-cat was determined by florescence enzyme immunoassay with capsulated cellular polymer solid-phase (ImmunoCAP) coupled with cat dander (REF 14.451201, Therm Fischer Sicentific Inc.) with the detection limit ranging from 0.1 to 100 Ku/L.The cut-off value for IgE positivity was considered 0.35 Ku/L.Circulating leptin levels were measured using a commercial ELISA kit following manufacturer's instructions (Enzo Life Sciences, Farmingdale, NY).Plates were read at 450 nm in ELISA reader (Dynex Technologies, USA).Lyophilized leptin ranging from 31.3-2000 pg/mL was used to construct the standard curve.The levels of different cytokines and IgE in the supernatants from cell cultures were determined using the "Th1/Th2/Th9/Th17 Cytokine 18-plex human Panel" kit (InvitroGen, San Diego, CA, USA) and human IgE ELISA kit (88-50610-22) (Invitrogen, Thermo Fisher Scientific Co), respectively.

Statistical analyses
All statistical analyses were conducted using the Prism 8.0 program (GraphPad Software).Comparisons between immune assays in non-stimulated (none) or activated PBMC cultures with Fel d1 and Fel d1/leptin were performed with one-way ANOVA, followed by Tukey test for data with Gaussian distribution, and by Kruskal-Wallis, followed by Dunn's test for data without Gaussian distribution.The nonparametric Mann-Whitney U test and Student's t test were applied to determine whether the two groups were statistically different for nonparametric and parametric variables, respectively.Pearson's and Spearman's correlation were applied for variables with or without normal distribution, respectively.Significance for all experiments was defined as p<0.05.

Leptin modulates cytokine and IgE production by Fel d1-stimulated PBMCs from cat-allergic patients
Table 1 shows that most cat allergic patients were overweight/ obese women who presented moderate or severe symptoms of rhinitis (AR) and asthma (AA).As no statistical difference among patients with different clinical symptoms (AR x AA x AR/AA) was observed with regard to immunological assays, they were all included together as a single patient group (CAP-cat allergic patients).For the control group, some experiments were additionally performed in 20 age-and gender-matched healthy subjects (HS).Higher levels of IL-4, IL-5, IL-6, IL-13 and IL-17 were observed in CAP-derived PBMC cultures containing plolyclonllyactivated T cells, as compared with HS (Figure S2).In those cell cultures, leptin elevated the release of IL-6, IFN-g and IL-17 in HS group and the secretion of IL-5, IL-6, IL-13 and IL-17 in CAP group.In contrast, this adipokine reduced the levels of IL-10 secreted by mitogen-activated T cells from both experimental groups (Figure S2).Concerning the cytokine profile in response to Fel d1, this major cat antigen induced not only the production of IL-4, IL-5, IL-13, IL-9, IL-6, IL-17, IL-21 and IL-10 (Figure 1A), but also the secretion of IgE (Figure 1B).The addition of leptin increased the release of IL-5, IL-13, IL-6, IL-17, IL-21 (Figure 1A) and IgE (Figure 1B), but reduced IL-10 production (Figure 1A).Of note, neither medium nor leptin alone were not able to induce detectable cytokine (data not shown).Concerning the control group, Fel d1 only significantly elevated the production of IL-10, with no difference after leptin addition (Figure S3).In patients, in vitro IgE production directly correlated with both IL-4 and IL-5, released by Fel d1-stimulated cells (Table 2), and IL-4, IL-5 and IL-9, secreted by Fel d1/Lep-activated PBMC cultures (Table 2).In contrast, in Fel d1/Lep-stimulated cells, IL-10 secretion inversely correlated to IgE (Table 2).
Leptin not only enhanced the proportion of T FH IL-21 + IL-5 + (Figure 2M) and T FH IL-21 + IL-9 + (Figure 2N), but also that of T FH IL-21 -cells positive for IL-4 (Figure 2L), IL-5 (Figure 2M), and IL-13 (Figure 2O).The ability of leptin to upregulate non-T FH and T FH cell phenotypes was observed in cell cultures from lean, overweight and obese patients (data not shown).

Correlation between plasma leptin levels, anti-cat IgE titers and the in vitro cytokine profile in CAP.
As demonstrated in Table 3, leptin levels positively correlated with IL-5, IL-6 and IL-17 secretion by Fel d1-stimulated cells, as well as the frequency of both non-T FH (IL-5 + , IL-13 + and IL-17 + ) and T FH IL-21 -cells positive for IL-5, IL-9 and IL-13.In contrast, a negative correlation was observed between circulating levels of this adipokine and the proportion of Treg and T FR cells, expressing or not CD39 marker.Moreover, the proportion of CD39 + Tr-1 cells inversely correlated with leptin concentration (Table 3).Although no significant correlation was observed between plasma leptin and anti-cat IgE (r=0.4054,p=0.0845), titers of this antibody positively correlated with both IL-5 release and the percentage of T FH IL-21 - IL-4 + and IL-21 -IL-13 + cells in Fel d1-stimulated PBMC cultures (Table 4).On the other hand, higher levels of this antibody were observed in patients with lower Treg and T FR cell proportions, expressing CD39 or not, and Tr-1 CD39 + cells (Table 4).

Discussion
Obesity can complicate IgE atopic diseases (23,34).Here, in cat allergic patients, this adverse relationship should involve, at least in part, increased leptin production that promotes an imbalance between different CD4 + T cell phenotypes specific to Fel d1, the major cat allergen.In the present study, Fel d1 not only increased the production of cytokines related to Th2 and Th9 cells, but also the proportion of antigen-specific T FH cell subsets, mainly IL-21 -IL-4 + , IL-21 -IL-5 + and IL-21 -IL-13 + .Despite the small sample size in this study, a higher percentage of Fel d1-specific Th2-like cells and T FH 2/T FH 13 cell phenotypes was observed among obese patients and directly correlated with plasma leptin levels.In vitro, this adipokine directly favored the expansion of Fel d1-specific Th2-and Th9-related phenotypes, as well as elevated the percentage of T FH IL-21 + (IL-5 + and IL-9 + ) and T FH IL-21 -(IL-4 + , IL-5 + and IL-13 + ) cell subsets.Additionally, leptin elevated IgE production.This finding agrees with a study that demonstrated a direct relationship between leptin levels and IgE production in atopic patients (25).Regarding cell phenotypes, IgE levels in PBMC cultures stimulated with Fel d1/ Lep directly correlated with the frequency of T FH IL-21 + IL-5 + and T HF IL-21 -negative for IL-4, IL-5, IL-13 and IL-9, but not Th2-like cells.Furthermore, plasma titers of anti-Fel d1 IgE positively correlated with T FH IL-21 -positive for IL-5 and IL-13.This finding agrees with studies that demonstrate that T FH cells, but not Th2 cells, are critical for IgE production (8, 17).Among T FH cells, while the T FH 2 cell subset induces the production of low-affinity IgE (8). the T FH 13 cell subset is responsible for producing high-affinity IgE (17).T FH 13 cells are characterized by high IL-4, IL-5 and IL-13 expression associated with very low IL-21 production (25).Yang et al. (35) demonstrated that IL-21 inhibits IgE class-switch recombination in human B cells.Although we did not evaluate either T FH cells that simultaneously express IL-4, IL-5 and IL-13, nor IgE affinity, we believe that the ability of leptin to increase the frequency of Fel d1specific T FH IL-21 -able to produce Th2-related cytokines is one of the mechanisms that this adipokine uses to intensify cat allergy severity.Indeed, the formation of high affinity IgE : FcϵRI complexes on mast cells, by activating Lyn/Syk/LAT-1 axis, promotes intense and immediate histamine release and leukotriene synthesis (18,35,36), resulting in associated allergic symptoms, such as airway constriction, increased mucus production, and coughing.
Despite not directly inducing IgE production, Th2 cytokines participate in the pathogenesis of atopic allergic reactions.IL-4 and IL-13 amplify eosinophil and Th2 cell transmigration to the allergen exposure site (37).IL-5 is responsible for increasing eosinophil formation and survival (38).IL-13 increases B cell survival (18) and compromises respiratory function by increasing mucus production in the airways (39).Therefore, leptin ability to increase the frequency of Fel d1-specific Th2-like cells should impact the severity of allergic reactions to cats.Indeed, leptin, by potentiating Th2-mediated response, has been associated with atopic diseases (40).Moreover, here, leptin also favored expansion of Fel d1-specific Th9-like cells.IL-9, along with IL-5 and IL-13, prolong mast cell and eosinophil survival, and increase mucus production (20, 41).Interestingly, despite the lack of data about human T FH 9 cells, in murine allergy models, these cells support memory IgE + B cell generation (42, 43).Finally, the ability of leptin to upregulate Fel d1-induced IL-6 production may also contribute to IgE synthesis, since IL-6 favors B-cell proliferation, plasma cell survival, and antibody production (44)(45)(46).
Recently, the severity of mite-allergic asthma has been associated with Der f3-specific Th17 cells (47).Furthermore, IL-17 directly promoted IgE production by human B cells (48) and favors eosinophil accumulation in mucosa of atopic patients (49).In the present study, although Fel d1 has induced T FH IL-17 + , and leptin amplified this cell subtype, no relationship was observed with either in vitro IgE production or plasma anti-Fel d1 IgE.However, it is possible that during disease exacerbation, this cell phenotype may    Regarding regulatory CD4 + T cells, the severity of allergic reactions has been associated with functional damage of allergen-specific Treg cells (CXCR5 -FoxP3 + IL-10 + ), Tr-1 (CXCR5 -FoxP3 -IL-10 + ) and, mainly in IgE-mediated disorders, T FR (CXCR5 + FoxP3 + IL-10 + ) (21,22).In atopic patients, severity of the symptoms correlated with both dysfunctional T FR cells and elevated frequency of T FH 2 cells highly capable of assisting IgE production by allergen-specific B cells (13, 32, 50, 51).Those atopic-derived T FR cells show impaired IL-10 production, a net anti-inflammatory cytokine (32, 50, 52).In addition to IL-10, several surface biomarkers can identify highly functional regulatory T cells, such as CD39 (53).In the present study, leptin reduced the frequency of Fel d1-specific Treg/Tr-1 and T FR cells, most of them expressing CD39.It is known that CD39, along with CD73, metabolizes the extracellular adenosine triphosphate (ATP)/adenosine diphosphate (ADP) into adenosine (ADO), a metabolite which inhibits pro-inflammatory T cell phenotypes (53).A study by Li et al. observed the role of CD39 + Treg cells in controlling airway inflammation in the murine model of allergic asthma (54).Notably, the frequency of Treg and T FR cells, expressing CD39 or not, inversely correlated with both IgE production in Fel d1/Lep-stimulated cell cultures and plasma anti-Fel d1 IgE titers.In agreement with our findings, a study by Martin-Orozco et al. (55), demonstrated an inverse relationship between FoxP3 expression in the regulatory CD4 + T cell compartment with serum IgE levels and eosinophilia.Therefore, in the present study, high dose of leptin should negatively impact the prognostic of allergic diseases due to the ability of this adipokine in reducing functional Treg/Tr-1 and T FR cells, CD4 T cell subset implicated in controlling Th2/Th9 and T FH 2/T FH 13 axis respectively (21,22).

Conclusions
Although preliminary, our findings suggest that hyperleptinemia, by favoring expansion of pathogenic Fel d1-specific CD4 + T cells and impairing the functioning of regulatory CD4 + T cell subsets, would not only exacerbate disease severity, but also negatively impacts the success of allergen-specific immunotherapies against cat allergies (56, 57).The author(s) declare that no financial support was received for the research, authorship, and/or publication of this article.This study was supported by the Fundacão de Amparo à Pesquisa Carlos Chagas Filho (FAPERJ, grant number: E-26/202.940/2017).

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FIGURE 1Leptin modulates the cytokine profile and IgE production by PBMCs from cat-allergic patients in response to Fel 1d.PBMC cultures (1 x 10 6 /mL) from cat-allergic patients (n=30) were maintained for 6 days in the presence of culture medium alone (without) or with 10 mg/mL of Fel 1d, with or without 50 ng/mL of leptin (Lep).At the end of the culture time, the supernatants were harvested and the (A) cytokine (IL-4, IL-5, IL-13, IL-6, IL-17, IL-21, IFN-g and IL-10) and (B) IgE levels were determined by Luminex and ELISA, respectively.Mean values were compared using one-way ANOVA and p values are shown in the graphs.All data are shown as mean ± SD of six independent experiments with 4 and 6 samples per experiment.
FIGURE 2Leptin effect on the frequency of different Fel d1-specific T FH and non-T FH cell subsets in cat-allergic patients.PBMCs (1 x 10 6 /mL) from cat-allergic patients (n=30) were cultured for 6 days in the presence of culture medium alone (none) or with 10 µg/mL Fel 1d, with or without 50 ng/mL leptin (Lep).At the end of the culture time, and adopting the gating strategy shown in graph (A), the mean ± SD of conventional CD4 + T cells (non-T FH , CXCR5 -) and total T FH cells (T FH , CXCR5+) (B), as well as the T FH PD-1 + cell subset (C) were analyzed by cytometry.In (D, K), representative dot-plots of cytokine-producing non-T FH and T FH cells were shown, respectively.In (E-J), the mean ± SD of percentage of (E) IL-4 + , (F) IL-5 + , (G) IL-9 + , (H) IL-13 + , (I) IL-17 + and (J) IL-21 + among non-T FH cells, while (L-P) showed the mean ± SD values for T FH IL-21 + and T FH IL-21 -cells able to produce IL-4 (L), IL-5 (M), IL-9 (N), IL-13 (O), and IL-17 (P).Data are shown as mean ± SD of five independent experiments with 2 and 6 samples per experiment.Significance was calculated using one-way ANOVA and p values are shown in the graphs.

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FIGURE 3Leptin reduced the frequency of Fel d1-specific Tregs and T FR cell subsets in PBMC cultures from cat-allergic patients.Taking into account the expression of CXCR5, FoxP3, IL-10 and CD39, and following representative dot-plots and histograms shown in graphs (A, D), the frequency of (A) conventional (Tregs/Tr-1, CXCR5 -) and (D) follicular (T FR /T FR -1, CXCR5 + ) regulatory CD4 + T cells was determined in PBMC cultures from catallergic patients (n=30) after stimulation for 6 days with Fel d1 and Fel d1/Lep.The mean values ( ± SD) of FoxP3 + IL-10 + CD39 -and FoxP3 + IL-10 + CD39 + (B, E), and FoxP3 -IL-10 + CD39 -and FoxP3 -IL-10 + CD39 + (C, F), on Fel D1-specific Treg and T FR cell subsets was determined.Data are shown as mean ± SD of five independent experiments with 2 and 6 samples per experiment.Significance was calculated using one-way ANOVA and p values are shown in the graphs.

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FIGURE 4The frequency of effector and regulatory Fel d1-specific CD4 + T cell subsets according BMI.In cat-allergic patients (n=30), the frequency of cytokine-producing non-T FH cells (A), IL-21 + (B) and IL-21 -T FH cells (C), as well as Treg/Tr-1 (D) and T FR (E) cells in response to Fel d1 was correlated with BMI by using Pearson's correlation.

TABLE 1
The characteristics of subjects.

TABLE 2
Correlation between in vitro total IgE production and cytokine profile in Fel d1-stimulated PBMC cultures from cat-allergic patients.

TABLE 2 Continued
In PBMC cultures from cat-allergic patients (n=30) stimulated with Fel d1 or Fel d1/Lep, the levels of cytokines, determined via Luminex, and the frequency of different effector and regulatory CD4 + T cells subsets, evaluated by cytometry, was correlated with the in vitro total IgE concentration, assayed through ELISA.Bold values indicate significance (p<0.05).

TABLE 3
Correlation between plasma leptin levels and cytokine profile of cells in Fel d1-stimlated PBMC cultures from cat-allergic patients.

TABLE 4
Correlation between total IgE titers and cytokine profiles of Fel d1-stimlated PBMC cultures from cat-allergic patients.

TABLE 4 Continued
IgE (Ku/L)The titer of plasma total IgE was correlated with both cytokines levels, evaluated by Luminex, and the frequency, determined by cytometry, of different conventional (non-T FH, CXCR5 -) and follicular helper (T FH , CXCR5 + ) CD4 + T cell subsets in Fel d1-stimulated PBMC cultures from cat-allergic patients (n=30).Bold values indicate significance (p<0.05).